Poster Title

Presenter Information

Grade Level at Time of Presentation

Senior

Institution

University of Louisville

KY House District #

60

KY Senate District #

11

Faculty ​Advisor/​ Mentor

Dr. David Hein

Department

Pharmacology & Toxicology

Abstract

Arylamine N-acetyltransferase 1 (NAT1) was originally discovered as a xenobiotic metabolizing enzyme. NAT1 is ubiquitously expressed in human tissue. The endogenous role of NAT1 in breast cancer piqued our interest due to high levels of expression of NAT1 in ER positive breast cancers. We investigated the MDA-MB-231 cell line, which has relatively lower NAT1 expression than its ER positive counterparts. MDA-MB-231 CRISPR/Cas9-mediated knockouts have been created with no detectable NAT1 activity. These cell lines showed a dramatic difference in the ability to form colonies in soft agar, which suggests that the absence of NAT1 affects anchorage-independent growth. Therefore we utilized the MDA-MB-231 CRISPR/Cas9 NAT1 knockouts in this investigation to test anchorage-independent growth and invasive abilities. We hypothesized that the knockouts would be susceptible to anchorage-dependent apoptosis (anoikis) and have a decreased invasive ability compared to the parental line. Both the CRISPR/Cas9-mediated NAT1 knockout and the parental line were compared side by side in anoikis assays spanning six days, Transwell® invasion, and migration assays. Albeit prior literature reported a decrease in invasive ability in vitro, we determined that the absence of NAT1 in MDA-MB-231 cell lines had little to no effect on anchorage-independent growth and the cellular invasion ability (P= 0.19). Thus, the absence of NAT1 in MDA-MB-231 cells did not affect anchorage-independence or invasion, but rather the ability of those cells to grow in close proximity to one another.

Research funding was supported by the University of Louisville and the National Cancer Institute R25-CA134283 grant.

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Arylamine N-acetyltransferase 1 (NAT1) was originally discovered as a xenobiotic metabolizing enzyme. NAT1 is ubiquitously expressed in human tissue. The endogenous role of NAT1 in breast cancer piqued our interest due to high levels of expression of NAT1 in ER positive breast cancers. We investigated the MDA-MB-231 cell line, which has relatively lower NAT1 expression than its ER positive counterparts. MDA-MB-231 CRISPR/Cas9-mediated knockouts have been created with no detectable NAT1 activity. These cell lines showed a dramatic difference in the ability to form colonies in soft agar, which suggests that the absence of NAT1 affects anchorage-independent growth. Therefore we utilized the MDA-MB-231 CRISPR/Cas9 NAT1 knockouts in this investigation to test anchorage-independent growth and invasive abilities. We hypothesized that the knockouts would be susceptible to anchorage-dependent apoptosis (anoikis) and have a decreased invasive ability compared to the parental line. Both the CRISPR/Cas9-mediated NAT1 knockout and the parental line were compared side by side in anoikis assays spanning six days, Transwell® invasion, and migration assays. Albeit prior literature reported a decrease in invasive ability in vitro, we determined that the absence of NAT1 in MDA-MB-231 cell lines had little to no effect on anchorage-independent growth and the cellular invasion ability (P= 0.19). Thus, the absence of NAT1 in MDA-MB-231 cells did not affect anchorage-independence or invasion, but rather the ability of those cells to grow in close proximity to one another.

Research funding was supported by the University of Louisville and the National Cancer Institute R25-CA134283 grant.